Team:USP-EEL-Brazil/Experiments
Experimentos
Molecular Biology
Some news this fine day!
Our part are constituted of RBS and gene. The promoter and the terminator were used of BBa_R0010 (Kit plate 3 Well 4G). The cloning was made inserting our part into the BBa_R0010. Our DNA was synthetized in gBlock format by IDT.(Colocar a foto que tem os tubinhos e a IDT)
Our synthetic DNA were synthetized in gBlocks. For our part to become functional we did Gibson Assembly to lligate the gBlocks.
(Colocar a foto de gel de agarose com duas bandas, escrito "pleurotus" e "phoma")
Confirmed the Gibson assembly we amplified the Gibson products by PCR.
(Colocar a foto de gel de agarose com 4 bandas, duas de cada tamanho)
Made the amplification of genes, we prepared the vector with LacI promoter (BBa_R0010) and linearized plasmide backbone pSB1C3 (2016) for insertion of genes. The BBa_R0010 was transformaded in DH5alpha for production of more plasmids.
(colocar a foto "Miniprep LacI promoter in pSB1C3 2270 pb")
With the plasmids and the our part ready, we did 3A Assembly to insert our gene. First, we inserted the gene into linearized plasmid pSB1C3 from USP-EEL BRAZIL Team 2016, because we do not have the DpnI enzyme. For this reason, we tried insert also into BBa_J04450.
Expression and Purification
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Biochemical Characterization
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